This disclosure relates to bacterial diagnostics and more particularly to the detection of carbapenemase resistant genes that cause carbapenemase-resistance in bacteria.
β-Lactam antibiotics (beta-lactam antibiotics) are a broad class of antibiotics, consisting of all antibiotic agents that contain a β-lactam ring in their molecular structures. This includes penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. (Holten K B, Onusko E M (August 2000)). Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics. β-Lactam antibiotics are indicated for the prophylaxis and treatment of bacterial infections caused by susceptible organisms. At first, β-lactam antibiotics were mainly active only against Gram-positive bacteria, yet the recent development of broad-spectrum β-lactam antibiotics active against various Gram-negative organisms has increased their usefulness.
Bacterial resistance to antibacterial drugs has been increasing relentlessly over the past two decades. This includes common residents of the human body: Staphylococcus aureus (methicillin resistant or MRSA) Enteroccus faecalis and E. faecium (vancomycin resistant or VRE): Enterobacteriaceae (multiresistant, carbapenems included or CRE). It also includes environmental, opportunistic, but intrinsically multiresistant species: Pseudomonas aeruginosa and Acinetobacter baumannii. (Georgopapadakou (2014)).
CRE, which stands for carbapenem-resistant Enterobacteriaceae, are a family of bacteria that are difficult to treat because they have high levels of resistance to antibiotics. Klebsiella species and Escherichia coli (E. coli) are examples of Enterobacteriaceae, a normal part of the human gut bacteria that can become carbapenem-resistant. Types of CRE are sometimes known as KPC (Klebsiella pneumoniae carbapenemase) and NDM-1 (New Delhi Metallo-beta-lactamase). KPC and NDM-1 are enzymes that break down carbapenems and make them ineffective. Both of these enzymes, as well as the enzyme VIM (Verona Integron-Mediated Metallo-β-lactamase) have also been reported in Pseudomonas. The most common CREs are KPC, VIM, NDM, IMP (IMP-type carbapenemase) and OXA 48. Ongoing global dissemination of blaOXA-48-like, as well as the coexistence of blaNDM-1 and blaOXA-181 in a single K. pneumoniae strain have been documented.
Primers and probes have previously been described for NDM1 (US20120129180A1, WO2012023054A2), KPC (U.S. Pat. No. 7,968,292, US20120129180A1, US20110190170A1 and WO 2013/078565), IMP (US20120129180A1, US 20090163382 and US20090317807), VIM (US 20090163382, US20120129180 and US20090317807) and OXA (US20090163382A1, US20120129180A1 and U.S. Pat. No. 6,905,848). Multiplex analysis by melting curve analysis using a single dye was reported in US Application NO: 20120129180A1. U.S. Pat. No. 8,124,382 discloses multiplex PCR of tem, shy, ctx-m-1, ctx-m-9, mox, cit, dha, ebc and fox. Amplified targets were detected with a universal-labeled TAMRA dye in array format. US Application 20090163382 discloses the multiplexing of multiple amplified targets with immobilized capture probes on microarrays, and fluorescence emitted from the microarrays was measured.
There exists a clinical need for the rapid detection of the carriers of antibiotic resistant carbapenem-resistant Enterobacteriaceae of the NDM1, KPC, IMP, VIM and OXA genes having higher clinical prevalence rates.